PCSK9 (proprotein convertase subtilisin/kexin type 9) plays an important role in the control of circulating LDL- cholesterol (LDL-C) levels via modulation of rates of degradation of hepatic LDL receptor (LDLR). One important aspect of the PCSK9-LDLR pathway in mediating LDL-C clearance is that their transcription is coordinately regulated by sterols through a common sterol regulatory element (SRE) motif embedded in their gene promoters and is co-induced by current cholesterol lowering drugs such as statins through activation of SRE binding proteins. Statin treatment increases the transcription of both LDLR and PCSK9. The increased PCSK9 transcription leads to elevated plasma PCSK9 levels, which, in turn, reduces the number of LDLR on the surface of hepatocytes. The undesirable stimulatory action of statins on PCSK9 transcription is increasingly recognized as a major limitation to the therapeutic efficacy of statins in lowering plasma LDL-C. Our laboratory has made a novel discovery that PCSK9 gene transcription is regulated by the hepatocyte nuclear factor 1? (HNF1?) through a highly conserved HNF1 binding site of the PCSK9 gene promoter, whereas HNF1? binding site is absent on the LDLR gene promoter. Furthermore, we have demonstrated that HNF1 site and its trans-activator HNF1? are prominently involved in the inhibition of PCSK9 transcription by the natural cholesterol-lowering compound berberine (BBR) via a mechanism that reduces the intracellular protein level of HNF1?. Recently, we have obtained new evidence indicating the potential involvement of ubiquitin-proteasome pathway in BBR induced down regulation of HNF1? protein. In addition, by applying a unique technique that combines the DNA affinity pull-down (biotinylated HNF1-PCSK9 oligonucleotide probe) with high resolution mass spectrometry, we have identified the nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) as a novel HNF1? interacting protein, implicating its potential role in the regulation of PCSK9 transcription and repression by BBR. Altogether, our new findings have demonstrated for the first time that PCSK9 and LDLR transcription can be separately regulated and also provided a molecular basis for developing novel therapeutic interventions to selectively repress PCSK9 expression without diminishing LDLR transcription for the treatment of hypercholesterolemia and heart disease. The overall goal of this revised proposal is to elucidate the molecular and cellular mechanisms by which BBR inhibits PCSK9 transcription through its actions on HNF1? protein stability and its association with coactivators in hepatic cell lines and animal models. The three specific aims are: 1) to characterize HNF1? coactivators which are specifically involved in PCSK9 transcription and regulation by BBR; 2) to elucidate the molecular mechanisms by which BBR inhibits PCSK9 transcription through the ubiquitin-proteasome pathway mediated reduction of HNF1? protein levels; and 3) to examine the effects of liver-specific depletion of HNF1? on PCSK9, LDLR, plasma LDL-C metabolism in response to treatment of hyperlipidemic animals with either statin or BBR. PUBLIC HEALTH RELEVANCE: The success of these proposed studies will ultimately benefit hypercholesterolemic patients for their individual needs in reducing plasma cholesterol levels to prevent atherosclerotic cardiovascular disease.